PROJECT SUMMARY Initiation of translation in eukaryotic organisms is a complex and regulated process. Canonical initiation involves a coordinated assembly of ribosomal 40S subunit bound with initiator tRNA and trimeric factor eIF2 on the 5'-end of messenger RNAs (mRNAs) containing a 5' cap structure. This process is guided by >10 initiation factors that mediate loading of 40S subunits, subsequent ATP-dependent scanning of the ribosomal complex through a 5' untranslated region until the first start codon is recognized, and finally 60S subunit joining to begin translation. Here, we will delineate the dynamic pathways of translation initiation using multicolor single- molecule fluorescence to track initiation directly in real time. Our approach will use the genetically, biochemically and structurally well-characterized yeast translation system to reveal the real-time composition and conformation of translation initiation complexes. In Aim 1, we will investigate the composition of initiation complexes on 4 model mRNAs that span distinct initiation mechanisms. In Aim 2, we will observe initiation on the same mRNAs using conformational signals monitored by FRET: conformation of the 40S subunit, mRNA, and factors, and their interrelation will be determined using single-molecule methods. In Aim 3, we will delineate the mechanistic pathways of ribosomal scanning through 5'-untranslated regions (UTRs) that differ in length, sequence and structure. We will determine the timing of scanning, and compare results for predicted timescales from different models of scanning; we will also monitor scanning directly, and determine the time-dependent composition of a scanning ribosomal complex. Our central hypothesis is that distinct dynamic branch points exist during initiation, leading to different potential pathways and timescales of initiation. The expected results should provide a dynamic overview of the complex process of initiation, aiding in our understanding of translation in basic biology and disease. RELEVANCE Translation is the endpoint of gene expression and highly regulated. Translational control allows rapid and spatially localized response to stimuli. Disruption of translational regulation has been implicated in many human diseases, such as cancers and neurodegenerative disease. Understanding of initiation mechanisms is essential for future therapeutic intervention.